We have analyzed the effect of insertion mutants between the SV40 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4-, 42- or 90-bp of DNA at the SV40 NcoI site (m.p. 37) have been analyzed for their effect on SV40 early expression and positioning of the RNA 5' ends. Insertion of 4-bp reduced SV40 early promoter dependent CAT expression by 6- to 8-fold. Increasing the size of the insertion to 42- or 90-bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42- or 90-bp of DNA resulted in a decrease in EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats and/or the 72-bp repeats. The fact that LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control.